PAD4 functions upstream from salicylic acid to control defense responses in Arabidopsis.

The Arabidopsis PAD4 gene was previously shown to be required for synthesis of camalexin in response to infection by the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 but not in response to challenge by the non-host fungal pathogen Cochliobolus carbonum. In this study, we show that pad4 mutants exhibit defects in defense responses, including camalexin synthesis and pathogenesis-related PR-1 gene expression, when infected by P. s. maculicola ES4 326. No such defects were observed in response to infection by an isogenic avirulent strain carrying the avirulence gene avrRpt2. In P. s. maculicola ES4 326-infected pad4 plants, synthesis of salicylic acid (SA) was found to be reduced and delayed when compared with SA synthesis in wild-type plants. Moreover, treatment of pad4 plants with SA partially reversed the camalexin deficiency and PR-1 gene expression phenotypes of P. s. maculicola ES4 326-infected pad4 plants. These findings support the hypothesis that PAD4 acts upstream from SA accumulation in regulating defense response expression in plants infected with P. s. maculicola ES4 326. A working model of the role of PAD4 in governing expression of defense responses is presented.     

Arabidopsis thaliana glutamate-cysteine ligase: functional properties, kinetic mechanism, and regulation of activity

In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione. Glutamatecysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Because the putative Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes, the identity of this protein as a GCL has been debated. We have purified AtGCL from an Escherichia coli expression system and demonstrated that the recombinant enzyme catalyzes the ATP-dependent formation of gamma-glutamylcysteine from glutamate (Km = 9.1 mm) and cysteine (Km = 2.7 mm). Glutathione feedback inhibits AtGCL (Ki approximately 1.0 mm). As with other GCL, buthionine sulfoximine and cystamine inactivate the Arabidopsis enzyme but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. The slower inactivation rates observed with AtGCL suggest that the active site differs structurally from that of other GCL. Global fitting analysis of initial velocity data indicates that a random terreactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consists of a catalytic subunit and a regulatory subunit, AtGCL functions and is regulated as a monomeric protein. In response to redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. These results explain the reported posttranslational change in AtGCL activity in response to oxidative stress.     

Non-host defense response in a novel Arabidopsis-Xanthomonas citri subsp. citri pathosystem

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive diseases of citrus. Progress of breeding citrus canker-resistant varieties is modest due to limited resistant germplasm resources and lack of candidate genes for genetic manipulation. The objective of this study is to establish a novel heterologous pathosystem between Xcc and the well-established model plant Arabidopsis thaliana for defense mechanism dissection and resistance gene identification. Our results indicate that Xcc bacteria neither grow nor decline in Arabidopsis, but induce multiple defense responses including callose deposition, reactive oxygen species and salicylic aicd (SA) production, and defense gene expression, indicating that Xcc activates non-host resistance in Arabidopsis. Moreover, Xcc-induced defense gene expression is suppressed or attenuated in several well-characterized SA signaling mutants including eds1, pad4, eds5, sid2, and npr1. Interestingly, resistance to Xcc is compromised only in eds1, pad4, and eds5, but not in sid2 and npr1. However, combining sid2 and npr1 in the sid2npr1 double mutant compromises resistance to Xcc, suggesting genetic interactions likely exist between SID2 and NPR1 in the non-host resistance against Xcc in Arabidopsis. These results demonstrate that the SA signaling pathway plays a critical role in regulating non-host defense against Xcc in Arabidopsis and suggest that the SA signaling pathway genes may hold great potential for breeding citrus canker-resistant varieties through modern gene transfer technology.     

Fumonisin B1-induced cell death in arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways

We have established an Arabidopsis protoplast model system to study plant cell death signaling. The fungal toxin fumonisin B1 (FB1) induces apoptosis-like programmed cell death (PCD) in wild-type protoplasts. FB1, however, only marginally affects the viability of protoplasts isolated from transgenic NahG plants, in which salicylic acid (SA) is metabolically degraded; from pad4-1 mutant plants, in which an SA amplification mechanism is thought to be impaired; or from jar1-1 or etr1-1 mutant plants, which are insensitive to jasmonate (JA) or ethylene (ET), respectively. FB1 susceptibility of wild-type protoplasts decreases in the dark, as does the cellular content of phenylalanine ammonia-lyase, a light-inducible enzyme involved in SA biosynthesis. Interestingly, however, FB1-induced PCD does not require the SA signal transmitter NPR1, given that npr1-1 protoplasts display wild-type FB1 susceptibility. Arabidopsis cpr1-1, cpr6-1, and acd2-2 protoplasts, in which the SA signaling pathway is constitutively activated, exhibit increased susceptibility to FB1. The cpr6-1 and acd2-2 mutants also constitutively express the JA and ET signaling pathways, but only the acd2-2 protoplasts undergo PCD in the absence of FB1. These results demonstrate that FB1 killing of Arabidopsis is light dependent and requires SA-, JA-, and ET-mediated signaling pathways as well as one or more unidentified factors activated by FB1 and the acd2-2 mutation.     

The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death

Salicylic acid (SA) has been proposed to antagonize jasmonic acid (JA) biosynthesis and signaling. We report, however, that in salicylate hydroxylase-expressing tobacco (Nicotiana tabacum) plants, where SA levels were reduced, JA levels were not elevated during a hypersensitive response elicited by Pseudomonas syringae pv phaseolicola. The effects of cotreatment with various concentrations of SA and JA were assessed in tobacco and Arabidopsis (Arabidopsis thaliana). These suggested that there was a transient synergistic enhancement in the expression of genes associated with either JA (PDF1.2 [defensin] and Thi1.2 [thionin]) or SA (PR1 [PR1a-beta-glucuronidase in tobacco]) signaling when both signals were applied at low (typically 10-100 microm) concentrations. Antagonism was observed at more prolonged treatment times or at higher concentrations. Similar results were also observed when adding the JA precursor, alpha-linolenic acid with SA. Synergic effects on gene expression and plant stress were NPR1- and COI1-dependent, SA- and JA-signaling components, respectively. Electrolyte leakage and Evans blue staining indicated that application of higher concentrations of SA + JA induced plant stress or death and elicited the generation of apoplastic reactive oxygen species. This was indicated by enhancement of hydrogen peroxide-responsive AoPR10-beta-glucuronidase expression, suppression of plant stress/death using catalase, and direct hydrogen peroxide measurements. Our data suggests that the outcomes of JA-SA interactions could be tailored to pathogen/pest attack by the relative concentration of each hormone.     

The Arabidopsis flavin-dependent monooxygenase FMO1 is an essential component of biologically induced systemic acquired resistance

Upon localized attack by necrotizing pathogens, plants gradually develop increased resistance against subsequent infections at the whole-plant level, a phenomenon known as systemic acquired resistance (SAR). To identify genes involved in the establishment of SAR, we pursued a strategy that combined gene expression information from microarray data with pathological characterization of selected Arabidopsis (Arabidopsis thaliana) T-DNA insertion lines. A gene that is up-regulated in Arabidopsis leaves inoculated with avirulent or virulent strains of the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) showed homology to flavin-dependent monooxygenases (FMO) and was designated as FMO1. An Arabidopsis knockout line of FMO1 proved to be fully impaired in the establishment of SAR triggered by avirulent (Psm avrRpm1) or virulent (Psm) bacteria. Loss of SAR in the fmo1 mutants was accompanied by the inability to initiate systemic accumulation of salicylic acid (SA) and systemic expression of diverse defense-related genes. In contrast, responses at the site of pathogen attack, including increases in the levels of the defense signals SA and jasmonic acid, camalexin accumulation, and expression of various defense genes, were induced in a similar manner in both fmo1 mutant and wild-type plants. Consistently, the fmo1 mutation did not significantly affect local disease resistance toward virulent or avirulent bacteria in naive plants. Induction of FMO1 expression at the site of pathogen inoculation is independent of SA signaling, but attenuated in the Arabidopsis eds1 and pad4 defense mutants. Importantly, FMO1 expression is also systemically induced upon localized P. syringae infection. This systemic up-regulation is missing in the SAR-defective SA pathway mutants sid2 and npr1, as well as in the defense mutant ndr1, indicating a close correlation between systemic FMO1 expression and SAR establishment. Our findings suggest that the presence of the FMO1 gene product in systemic tissue is critical for the development of SAR, possibly by synthesis of a metabolite required for the transduction or amplification of a signal during the early phases of SAR establishment in systemic leaves.     

Constitutive salicylic acid-dependent signaling in cpr1 and cpr6 mutants requires PAD4

Salicylic acid (SA)-dependent signaling controls activation of a set of plant defense mechanisms that are important for resistance to a variety of microbial pathogens. Many Arabidopsis mutants that display altered SA-dependent signaling have been isolated. We used double mutant analysis to determine the relative positions of the pad4, cpr1, cpr5, cpr6, dnd1 and dnd2 mutations in the signal transduction network leading to SA-dependent activation of defense gene expression and disease resistance. The pad4 mutation causes failure of SA accumulation in response to infection by certain pathogens, while the other mutations cause constitutively high levels of SA, defense gene expression and resistance. The cpr1 pad4, cpr5 pad4, cpr6 pad4, dnd1 pad4 and dnd2 pad4 double mutants were constructed and assayed for stature, presence of spontaneous lesions, resistance to Pseudomonas syringae and Peronospora parasitica, SA levels, expression of PAD4, PR-1 and PDF1.2, and accumulation of camalexin. We found that the effects of the cpr1 and cpr6 mutations on SA-dependent gene expression are completely dependent on PAD4 function. In contrast, SA accumulation in the lesion-mimic mutant cpr5 is partially PAD4-independent, while in dnd1 and dnd2 mutants it is completely PAD4-independent. A model describing a possible arrangement of activities in the signal transduction network is presented.     

Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits

Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and, in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic subunit (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate-limiting step in glutathione production. Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of these mutations on enzymatic function in vivo and in vitro to gain further insight into the pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC, and cellular glutathione levels were determined. The four mutant transfectants each had significantly lower levels of glutathione relative to that of the wild type, with the Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL activity were investigated using a Saccharomyces cerevisiae model system. Mutant GCLC alone could not complement a glutathione deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic constants comparable to those of wild-type GCLC, suggesting that heterodimer formation is needed for stability in vivo. Strategies that promote heterodimer formation and persistence would be effective therapeutics for the treatment of GCL deficiency.     

The glutathione-deficient mutant pad2-1 accumulates lower amounts of glucosinolates and is more susceptible to the insect herbivore Spodoptera littoralis

Plants often respond to pathogen or insect attack by inducing the synthesis of toxic compounds such as phytoalexins and glucosinolates (GS). The Arabidopsis mutant pad2-1 has reduced levels of the phytoalexin camalexin and is known for its increased susceptibility to fungal and bacterial pathogens. We found that pad2-1 is also more susceptible to the generalist insect Spodoptera littoralis but not to the specialist Pieris brassicae . The PAD2 gene encodes a γ-glutamylcysteine synthetase that is involved in glutathione (GSH) synthesis, and consequently the pad2-1 mutant contains about 20% of the GSH found in wild-type plants. Lower GSH levels of pad2-1 were correlated with reduced accumulation of the two major indole and aliphatic GSs of Arabidopsis, indolyl-3-methyl-GS and 4-methylsulfinylbutyl-GS, in response to insect feeding. This effect was specific to GSH, was not complemented by treatment of pad2-1 with the strong reducing agent dithiothreitol, and was not observed with the ascorbate-deficient mutant vtc1-1 . In contrast to the jasmonate-insensitive mutant coi1-1 , expression of insect-regulated and GS biosynthesis genes was not affected in pad2-1 . Our data suggest a crucial role for GSH in GS biosynthesis and insect resistance.     

Arabidopsis thaliana EDS4 contributes to salicylic acid (SA)-dependent expression of defense responses: evidence for inhibition of jasmonic acid signaling by SA

The Arabidopsis enhanced disease susceptibility 4 (eds4) mutation causes enhanced susceptibility to infection by the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326). Gene-for-gene resistance to bacteria carrying the avirulence gene avrRpt2 is not significantly affected by eds4. Plants homozygous for eds4 exhibit reduced expression of the pathogenesis-related gene PR-1 after infection by Psm ES4326, weakened responses to treatment with the signal molecule salicylic acid (SA), impairment of the systemic acquired resistance response, and reduced accumulation of SA after infection with Psm ES4326. These phenotypes indicate that EDS4 plays a role in SA-dependent signaling. SA has been shown to have a negative effect on activation of gene expression by the signal molecule jasmonic acid (JA). Two mutations that cause reduced SA levels, eds4 and pad4, cause heightened responses to inducers of JA-dependent gene expression, providing genetic evidence to support the idea that SA interferes with JA-dependent signaling. Two possible working models of the role of EDS4 in governing activation of defense responses are presented.     

ZINC TOLERANCE INDUCED BY IRON 1 reveals the importance of glutathione in the cross-homeostasis between zinc and iron in Arabidopsis thaliana

Zinc is an essential micronutrient for plants, but it is toxic in excess concentrations. In Arabidopsis, additional iron (Fe) can increase Zn tolerance. We isolated a mutant, zinc tolerance induced by iron 1, designated zir1, with a defect in Fe-mediated Zn tolerance. Using map-based cloning and genetic complementation, we identified that zir1 has a mutation of glutamate to lysine at position 385 on γ-glutamylcysteine synthetase (GSH1), the enzyme involved in glutathione biosynthesis. The zir1 mutant contains only 15% of the wild-type glutathione level. Blocking glutathione biosynthesis in wild-type plants by a specific inhibitor of GSH1, buthionine sulfoximine, resulted in loss of Fe-mediated Zn tolerance, which provides further evidence that glutathione plays an essential role in Fe-mediated Zn tolerance. Two glutathione-deficient mutant alleles of GSH1, pad2-1 and cad2-1, which contain 22% and 39%, respectively, of the wild-type glutathione level, revealed that a minimal glutathione level between 22 and 39% of the wild-type level is required for Fe-mediated Zn tolerance. Under excess Zn and Fe, the recovery of shoot Fe contents in pad2-1 and cad2-1 was lower than that of the wild type. However, the phytochelatin-deficient mutant cad1-3 showed normal Fe-mediated Zn tolerance. These results indicate a specific role of glutathione in Fe-mediated Zn tolerance. The induced accumulation of glutathione in response to excess Zn and Fe suggests that glutathione plays a specific role in Fe-mediated Zn tolerance in Arabidopsis. We conclude that glutathione is required for the cross-homeostasis between Zn and Fe in Arabidopsis.